The experiments proposed herein will explore new models for effects of WNK kinases, kelch-like 3, and cullin 3 on the thiazide-sensitive Na-Cl cotransporter (NCC). The NCC of the mammalian kidney is essential to normal blood pressure regulation and to normal potassium balance. Mutations in the transporter cause salt wasting and hypokalemia. Mutations in the WNK kinases, kelch-like 3 or cullin 3 lead to hypertension and hyperkalemia by activating the transporter. Despite intense efforts, mechanisms by which these mutations cause human disease remain unclear. We have recently developed two novel mouse models, permitting us to study how the WNK kinases and cullin 3 signal to the thiazide-sensitive transporter, in vivo. These models compel us to propose novel, paradigm-shifting models for WNK and cullin 3 signaling to NCC. Here, we propose to determine how WNK4 can act both as an inhibitor and a stimulator of ion transport in the distal nephron. We will also test how interactions with other members of the WNK kinase family help to explain the sometimes-contradictory results obtained in the literature. We will also test a new model for cullin 3 regulation in NCC, one in which cullin 3 feeds back to regulate kelch-like 3, giving cullin 3 both stimulatory and inhibitory effects on NCC. To accomplish these goals, we have recruited three prominent research groups from around the world to work together. We will take the models generated using in vivo approaches, and explore the mechanisms involved using well-established in vitro approaches. To this end, we will 1) Determine how the WNK signaling complex formation modulates NCC activity, and 2) Determine how CRL activity modulates WNK kinase signaling. These aims will be performed using both in vivo and in vitro techniques, insuring that conclusions are physiologically sound and relevant. The long-term goal is to determine how balance and sodium balance can be separated pharmacologically. Mutations in WNK kinases, kelch-like 3 and cullin 3 appear to dissociate the potassium-wasting effects of aldosterone from the sodium- retaining effects. We hope to learn how the kidney normally recognizes whether to excrete potassium or reabsorb salt, so that we can develop dietary better approaches to prevent hypertension, and better pharmaceutical approaches to treat it.